Protein-protein interactions are key molecular events that integrate multiple gene products into functional complexes in virtually every cellular process. These cellular processes are mostly mediated by non-covalently interacting multi-protein complexes and can include, for example, transcription, translation, metabolic pathways or signal transduction pathways. Because such interactions mediate numerous disease states and biological mechanisms underlying the pathogenesis of bacterial and viral infections, identification of protein-protein interactions remains one of the most important challenges in the post-genomics era. The most widely used is the yeast two-hybrid assay (Y2H), which was developed in 1989 (Fields et al., “A Novel Genetic System to Detect Protein-protein Interactions,” Nature 340; 245-246 (1989)). Briefly, this assay comprises two proteins fused to a split yeast transcription factor (originally GAL4) that binds a promoter upstream of a reporter protein. If the proteins interact, the activity of the transcription factor is reconstituted and transcription of the reporter protein is upregulated, providing a signal.
The yeast 2-hybrid (Y2H) system has been the tool of choice for revealing numerous protein-protein interactions that underly diverse protein networks and complex protein machinery inside living cells. Y2H assay has been used to generate protein interaction maps for humans (Rual J. F., et al., “Towards a Proteome-Scale Map of the Human Protein-protein Interaction Network,” Nature 437:1173-1178 (2005)). Another important application of the Y2H methodology is the discovery of diagnostic and therapeutic proteins, whose mode of action is high-affinity binding to a target peptide or protein. For example, several groups have isolated antibody fragments that are readily expressed in the cytoplasm of cells where they bind specifically to a desired target (der Maur et al., “Direct in vivo Screening of Intrabody Libraries Constructed on a Highly Stable Single-chain Framework,” J Biol Chem 277:45075-45085 (2002), Visintin M., et al., “Selection of Antibodies for Intracellular Function Using a Two-Hybrid in vivo System,” Proc Natl Acad Sci USA 96:11723-11728 (1999)), and in certain instances ablate protein function (Tanaka T. et al., “Intrabodies Based on Intracellular Capture Frameworks that Bind the RAS Protein with High Affinity and Impair Oncogenic Transformation,” EMBO J. 22:1025-1035 (2003), Tse E., et al., “Intracellular Antibody Capture Technology: Application to Selection of Intracellular Antibodies Recognising the BCR-ABL Oncogenic Protein,” J Mol Biol 31 7:85-94 (2002)). The yeast two-hybrid assay is highly versatile and is still widely used for analysis of the complex interactions of eukaryotic cellular networks. However, it has several drawbacks, including in the fact that it requires the nuclear environment of the eukaryotic yeast host, which may differ from the interaction environment of the proteins of interest.
The Y2H system was initially developed by using yeast as a host organism. Numerous bacterial (B)2H systems are now common laboratory tools and represent an experimental alternative with certain advantages over the yeast-based systems (Hu J. C. et al., “Escherichia coli One- and Two-hybrid Systems for the Analysis and Identification of Protein-protein Interactions,” Methods 20:80-94 (2000), Ladant D. et al., “Genetic Systems for Analyzing Protein-protein Interactions in Bacteria,” Res Microbiol 151:711-720 (2000)). A number of these bacterial approaches employ split activator/repressor proteins; thus, they are functionally analogous to the GAL4-based yeast system (Dove S. L. et al., “Activation of Prokaryotic Transcription Through Arbitrary Protein-protein Contacts,” Nature 386:627-630 (1997), Hu J. C., et al., “Sequence Requirements for Coiled-coils: Analysis with lambda Repressor-GCN4 Leucine Zipper Fusions,” Science 250:1400-1403 (1990), Joung J. K., et al., “A Bacterial Two-hybrid Selection System for Studying Protein-DNA and Protein-protein Interactions,” Proc Natl Acad Sci USA 97:7382-7387 (2000)). Unfortunately, both Y2H and B2H GAL4-type assays are prone to a high frequency of false positives that arise from spurious transcriptional activation (Fields S., “High-throughput two-hybrid Analysis. The Promise and the Peril,” FEBS J 272: 5391-5399 (2005)), and complicate the interpretation of interaction data. As proof, a comparative assessment revealed that >50% of the data generated using Y2H were likely to be false positives (von Mering C., et al., “Comparative Assessment of Large-scale Data Sets of Protein-protein Interactions,” Nature 41 7:399-403 (2002)). To address this shortcoming, several groups have exploited oligomerization assisted reassembly of split enzymes such as adenylate cyclase (Karimova G., et al., “A Bacterial Two-hybrid System Based on a Reconstituted Signal Transduction Pathway,” Proc Natl Acad Sci USA 95:5752-5756 (1998)), β-lactamase (Bla) (Wehrman T., et al., “Protein-protein Interactions Monitored in Mammalian Cells via Complementation of Beta-lactamase Enzyme Fragments,” Proc Natl Acad Sci USA 99:3469-3474 (2002)), and dihydrofolate reductase (Pelletier J. N., et al., “An in vivo Library-versus-library Selection of Optimized Protein-protein Interactions,” Nat Biotech 17:683-690 (1999), Pelletier J. N., et al., “Oligomerization Domain-directed Reassembly of Active Dihydrofolate Reductase from Rationally Designed Fragments,” Proc Natl Acad Sci USA 95:12141-12146 (1998)), as well as split fluorescent proteins (Ghosh I. et al., “Antiparallel Leucine Zipper-Directed Protein Reassembly: Application to the Green Fluorescent Protein,” J Am Chem Soc 122:5658-5659 (2000)). Alternatively, a number of methodologies for detecting interacting proteins in bacteria have been developed that do not rely on interaction-induced complementation of protein fragments, but instead use phage display (Palzkill T. et al., “Mapping Protein-ligand Interactions Using Whole Genome Phage Display Libraries,” Gene 221:79-83 (1998)), FRET (You X., et al., “Intracellular Protein Interaction Mapping with FRET Hybrids,” Proc Natl Acad Sci USA 103:18458-18463 (2006)), and cytolocalization of GFP (Ding Z., et al., “A Novel Cytology-based, Two-hybrid Screen for Bacteria Applied to Protein-protein Interaction Studies of a Type IV Secretion System,” J Bacteriol 184:5572-5582 (2002)).
In recent years, an alternative to the yeast two-hybrid assay has arisen in the form of the protein complementation assay (PCA). This method fuses the proteins of interest to a split reporter protein such as GFP (Cabantous, S. et al., “Recent Advances in GFP Folding Reporter and Split-GFP Solubility Reporter Technologies. Application to Improving the Folding and Solubility of Recalcitrant Proteins from Mycobacterium Tuberculosis,” J Struct Funct Genomics 6:113-119 (2005), Cabantous, S. et al., “In vivo and in vitro Protein Solubility Assays Using Split GFP,” Nat Methods 3:845-54 (2006)), YFP (Bracha-Drori, K. et al., “Detection of Protein-protein Interactions in Plants Using Bimolecular Fluorescence Complementation,” Plant J 40:419-427 (2004)), luciferase (Kim, S. B. et al., “High-throughput Sensing and Noninvasive Imaging of Protein Nuclear Transport by Using Reconstitution of Split Renilla Luciferase,” Proc Natl Acad Sci USA 101:11542-11547 (2004)), dihydrofolate reductase (Remy, I. et al., “Detection of Protein-protein Interactions Using a Simple Survival Protein-fragment Complementation Assay Based on the Enzyme Dihydrofolate Reductase,” Nat Proto 2:2120-2125 (2007)), or β-lactamase (Galarneau, A. et al., “Beta-lactamase Protein Fragment Complementation Assays as in vivo and in vitro Sensors of Protein-protein Interactions,” Nat Biotechnol 20:619-22 (2002), Wehrman, T. et al., “Protein-protein Interactions Monitored in Mammalian Cells via Complementation of Beta-lactamase Enzyme Fragments,” Proc Natl Acad Sci USA 99:3469-3474 (2002)). Using protein engineering techniques, split proteins whose individual fragments are inactive can be developed. Upon interaction of the proteins of interest, the split reporter protein regains its activity. Versions of a split β-lactamase (Bla) protein complementation assay for monitoring protein-protein interactions in mammalian cells have been developed (Galarneau, A. et al., “Beta-lactamase Protein Fragment Complementation Assays as in vivo and in vitro Sensors of Protein-protein Interactions,” Nat Biotechnol 20:619-22 (2002), Wehrman, T. et al., “Protein-protein Interactions Monitored in Mammalian Cells via Complementation of Beta-lactamase Enzyme Fragments,” Proc Natl Acad Sci USA 99:3469-3474 (2002)). These versions however contain no signal sequence for protein transport, and thus the proteins were expressed in the cytoplasm of E. coli. Following expression, the Bla (β-lactamase) activity was measured in vitro by nitrocefin colorimetric assay. One limitation of cytoplasmic expression is that cytoplasmic β-lactamase (Bla) is incapable of conferring resistance to ampicillin and thus genetic selection is not possible. In one version, Wehrman et al. employed prototypical Sec signal peptides for delivery of the fragments into the periplasm by the post-translational Sec export pathway (Wehrman, T. et al., “Protein-protein Interactions Monitored in Mammalian Cells via Complementation of Beta-lactamase Enzyme Fragments,” Proc Natl Acad Sci USA 99:3469-3474 (2002)). Following export, both fragments localize to the periplasm and cells can be selected on ampicillin. The limitation of this approach is that it uses post-translational Sec export signals. The proteins under investigation may fold fully inside the cytoplasm thereby limiting their potential translocation across the cytoplasmic membrane. They may also interact in the cytoplasm prior to export and thus not be exported to the periplasm. A further limitation of this approach is the fact that only one pair of small peptides was tested for interaction; whether split Bla (β-Lactamase) could be used to report the interactions between globular proteins was not demonstrated. Thus, to date there have been no reports detailing the use of split protein fragments for monitoring protein-protein interactions in the bacterial periplasm.
The present invention is directed to overcoming these and other deficiencies in the art.